Methods and compositions for treatment of macular and retinal disease

ABSTRACT

The present invention describes linking a therapeutic agent to a compound which is known to be naturally concentrated in a tissue affected by, or that is causing, a disease, to create a prodrug for treatment of the disease. Embodiments of the present invention include a new class of carotenoid-linked drugs to treat such blinding retinal disease such as age-related macular degeneration, retinoblastoma, and diabetic macular edema. For example, the present invention comprises a method for the treatment of a disorder of the eye comprising linking a therapeutic agent to a xanthophyll carotenoid to create a prodrug, and administering a therapeutically effective amount of the prodrug to an individual in need of treatment. Provided are prodrugs for treatment of retinoblastoma, cystoid macular edema (CME), exudative age-related macular degeneration (AMD), diabetic retinopathy, diabetic macular edema, or inflammatory disorders.

RELATED APPLICATIONS

This application claims priority to provisional application Ser. No.60/403,499, filed Aug. 14, 2002, and is a continuation application ofnon-provisional application Ser. No. 10/639,972, filed Aug. 13, 2003,now U.S. Pat. No. 7,259,180. The entire disclosures of provisionalapplication 60/403,499 and nonprovisional application Ser. No.10/639,972 are incorporated in its entirety herein.

FIELD OF THE INVENTION

The present invention relates to methods and compositions for treatmentand prevention of disease, and in particular embodiments, disorders ofthe eye. In an embodiment, the present invention describes linkingtherapeutic compounds to xanthophyll carotenoids to enhance accumulationof the therapeutic compound in the retina and macula of the eye.

BACKGROUND OF THE INVENTION

The retina is the part of the eye that is sensitive to light. The maculalutea is the region of the retina that allows us to read and recognizefaces. Diseases of the macula, such as age-related macular degenerationand diabetic macular edema, account for a major proportion of legalblindness. To combat these diseases, a variety of accepted andexperimental medications are employed via systemic routes or local,invasive surgical procedures.

A remarkable increase in knowledge and interest surrounding thepharmacologic treatment of macular and retinal diseases has occurred. Asa result, a variety of promising agents are now being investigated fortheir effects on such blinding disorders as exudative age-relatedmacular degeneration, diabetic retinopathy, macular edema,retinoblastoma, and other diseases of the retina and macula lutea.Currently, these drugs are delivered to the macula and retina via local,invasive surgical procedures, such as intravitreal or periorbitalinjections, or via systemic routes. Surgical methods often requirerepeated injections and may lead to serious ocular complications,including endophthalmitis, retinal detachment, and vitreous hemorrhage.Likewise, systemic administration is associated with a variety ofpotential systemic side effects and with the difficulty of deliveringtherapeutic levels of the drugs to the retina.

Given the recent advances in developing pharmacologic treatments for avariety of macular and retinal diseases, improving drug targeting anddelivery are of paramount importance. For example, retinoblastoma, aneoplastic process that arises from the retina, is the most commonprimary ocular cancer of childhood. Though once treated almostexclusively with radiation or eye removal (enucleation), systemicchemotherapy is now the sight and lifesaving therapy of choice. Inaddition, various anti-inflammatory and anti-angiogenic agents are beinginvestigated for use in treating exudative age-related maculardegeneration (AMD) (the leading cause of legal blindness in the elderly)and for diabetic retinopathy (the leading cause of blindness in workingAmericans). In order to advance the noninvasive, pharmacologic treatmentof retinoblastoma and other retinal diseases, new drugs having improveddelivery to the retina are needed.

SUMMARY

To advance the noninvasive, pharmacologic treatment of maculardegeneration, diabetic retinopathy, and other retinal diseases, thepresent invention describes the synthesis of a new class of drugscomprising a therapeutic agent known to be effective against aparticular eye disorder chemically linked to a carotenoid. Since themacula preferentially concentrates xanthophyll carotenoids within itslayers, xanthophyll carotenoids provide an ideal carrier for drugdelivery to the macula. In an embodiment, enhanced accumulation oftherapeutic medications using the conjugates of the present inventionconstitutes an improvement in drug efficacy over that achieved bycurrent routes of administration and to overall improved treatment ofmacular diseases.

Thus, in an embodiment, the present invention comprises a compound forthe treatment of an eye disorder comprising a therapeutic agent linkedto a carotenoid. In an embodiment, the carotenoid comprises axanthophyll carotenoid.

In another embodiment, the present invention comprises composition forthe treatment of an eye disorder comprising a pharmaceutically effectiveamount of a prodrug comprising a therapeutic agent linked to acarotenoid, and a pharmaceutically acceptable carrier, wherein apharmaceutically effective amount of the prodrug comprises an amountsufficient to ameliorate, prevent, or cure the eye disorder.

In yet another embodiment, the present invention describes a method forthe treatment of a disorder of the eye comprising linking a therapeuticagent to a carotenoid to create a prodrug, and administering apharmaceutically effective amount of the prodrug to an individual inneed of treatment, wherein a pharmaceutically effective amount of theprodrug comprises an amount sufficient to ameliorate, prevent or curesaid eye disorder.

In yet another embodiment, the present invention comprises a kit for thetreatment of an eye disorder comprising:

(a) a prodrug compound comprising a therapeutic agent linked to acarotenoid;

(b) a pharmaceutically acceptable carrier; and

(c) instructions to dispense a pharmaceutically effective amount of theprodrug mixed with the pharmaceutically acceptable carrier to anindividual in need thereof, wherein a pharmaceutically effective amountof the prodrug comprises an amount sufficient to ameliorate, prevent, orcure an eye disorder in the individual.

In an embodiment, the methods and compounds of the present invention mayalso used to treat diseases of other tissues known to concentratecarotenoids. For example, the compounds may also be used to treatdiseases of the liver or fat tissue.

Thus, in another embodiment, the present invention comprises a compoundfor the treatment of a disorder in a tissue type that is known toconcentrate carotenoids, comprising a therapeutic agent linked to acarotenoid.

In yet another embodiment, the present invention comprises a method forthe treatment of a disease comprising linking a therapeutic agent to acompound which is known to be naturally concentrated in a tissueaffected by, or that is causing, the disease to create a prodrug, andadministering a pharmaceutically effective amount of the prodrug to anindividual in need of treatment, wherein a pharmaceutically effectiveamount of the prodrug comprises an amount sufficient to ameliorate,prevent, or cure the disease.

For example, the present invention includes the synthesis of compoundshaving a xanthophyll carotenoid linked to therapeutic agents including,but not limited to, non-steroidal anti-inflammatory drugs (NSAIDs),steroids, anti-angiogenic drugs, anti-neoplastic agents, and drugs toprevent infectious disease (antiviral agents, antibacterial agents,anti-protozoan agents).

Thus, embodiments of the present invention describe the use of compoundswhich are naturally concentrated in the macula and retina to delivertherapeutic agents to these regions of the eye. Advantages associatedwith embodiments of the present invention include enhanced targeting ofvarious therapeutic medications through noninvasive means. Thus, oneadvantage associated with embodiments of the present invention includesincreased specificity and efficacy of drug treatment. Yet anotheradvantage associated with embodiments of the present invention is thatthe treatment results in fewer side effects and complications of due tothe targeted nature of the delivery system. Yet another advantageassociated with embodiments of the present invention is the ability toemploy systemic drug administration, as opposed to local, invasivesurgical procedures.

From the foregoing summary, it is apparent that an object of the presentinvention is to develop methods and compositions that will be effectivein targeting therapeutic agents to treat diseases of specific tissuetypes or organs. It is to be understood that the invention is notlimited in its application to the specific details as set forth in thefollowing description and figures. The invention is capable of otherembodiments and of being practiced or carried out in various ways.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the chemical structures of (3R,3′R,6′R)-lutein (Panel A),and (3R,3′R)-zeaxanthin (Panel B), which in accordance with anembodiment of the present invention, comprise xanthophyll carotenoidsthat may be linked to therapeutic agents to promote uptake of thetherapeutic agent in the eye.

FIG. 2 shows a schematic illustrating the formation of prodrugs forconcentrating therapeutics in the retina and macula of the eye havingeither a bipartate structure, with the therapeutic agent A₁ directlybound to the xanthophyll carrier (X—OH) (Panel A), or a tripartatestructure, with the therapeutic agent A₂ bound to the xanthophyllcarrier (X—OH) via an amino acid spacer (Panel B), in accordance with anembodiment of the present invention.

DETAILED DESCRIPTION

The present invention describes a new class of xanthophyll-linked drugsthat effectively treat such blinding retinal diseases as age-relatedmacular degeneration, retinoblastoma, diabetic macular edema andretinopathy. With advances in pharmacologic interventions for thesediseases, the xanthophyll-mediated drug delivery system of the presentinvention provides ocular specificity and minimal systemic side effects.

Thus, in one embodiment, the present invention comprises a compound forthe treatment of an eye disorder comprising a therapeutic agent linkedto a carotenoid. In an embodiment, the carotenoid comprises axanthophyll carotenoid.

As used herein, a therapeutic agent is a compound that has the abilityto ameliorate, prevent, or cure a particular disease. Also as usedherein, a carotenoids are lipophilic pigments having absorption peaksranging from 450-480 nm, and that are red, orange or yellow in color.Carotenoids include carotenes and xanthophylls. Also as used herein, acarotene comprises a hydrocarbon carotenoid, and a xanthophyll comprisesan oxygenated carotene.

In another embodiment, the present invention comprises a composition forthe treatment of an eye disorder comprising a pharmaceutically effectiveamount of a prodrug comprising a therapeutic agent linked to acarotenoid, and a pharmaceutically acceptable carrier, wherein apharmaceutically effective amount of the prodrug comprises an amountsufficient to ameliorate, prevent, or cure the eye disorder. In anembodiment, the carotenoid comprises a xanthophyll carotenoid.

Preferably, the carotenoid of the compounds and the compositions of thepresent invention acts as a carrier to facilitate systemic delivery ofthe therapeutic agent to the retina and/or the macula. As used herein, acarrier is a compound that facilitates delivery of a particular agent(e.g., a therapeutic drug) to the target area or tissue of interest. Acarrier may be covalently linked to the agent, as in the case of acarotenoid carrier which is linked to the therapeutic compound.Alternatively, the agent may be physically mixed with, or dissolved in,the carrier, as in the case where prodrugs of the present invention aredissolved in a physiological carrier (such as saline) for systemicinjection.

In an embodiment, the xanthophyll carotenoid comprises zeaxanthin.Alternatively, the xanthophyll may comprise lutein. Other xanthophyllsknown in the art, such as oxidation products of lutein and zeaxanthin,may be used. Thus, other xanthophyll compounds that may be used include,but are not limited to, 3′-epilutein, meso-zeaxanthin, 3-hydroxy-beta,epsilon-caroten-3′-one, epsilon-lycopenes, or 5-Z-lycopenes.

In an embodiment, the therapeutic agent used for the compounds and thecompositions of the present invention comprises an anti-angiogenicagent. As used herein, an anti-angiogenic agent comprises an agent thatameliorates or prevents vascularization of a tissue by the developmentof new capillary blood vessels. Preferably, the anti-angiogenic agentcomprises anecortave acetate, anti-VEGF aptamer (and other anti-VEGFagents), AMD-FAB, or protein kinase c inhibitor. Other anti-angiogenicagents known in the art may be used. Thus, other anti-angiogenic agentsthat may be used include, but are not limited to, steroids andangiostatic steroids, metalloproteinase inhibitors, and interferons.

Also, the therapeutic agent may comprise an anti-neoplastic agent. Asused herein, an anti-neoplastic agent comprises a compound that may beused to retard tumor or endothelial cell formation, growth, or spread.Preferably, the anti-neoplastic agent comprises etoposide orvincristine, which are anti-neoplastic agents used to treatretinoblastoma. Other anti-neoplastic agents known in the art may alsobe used.

In yet another embodiment, the therapeutic agent used for the compoundsand the compositions of the present invention comprises ananti-inflammatory agent. Preferably, the anti-inflammatory agentcomprises a steroid or a non-steroidal anti-inflammatory drug (NSAID).More preferably, the anti-inflammatory agent comprises ketorolac,triamcinolone or fluocinolone. Other anti-inflammatory agents known tobe effective in treating eye disease may be also be used. Thus, otheranti-inflammatory agents that may be used include, but are not limitedto, other steroids, interleukins, anti-leukotriene agents,cyclooxygenase inhibitors, and prostaglandin inhibitors.

In yet another embodiment, the therapeutic agent of the compounds andthe compositions of the present invention comprises agents to preventinfectious disease. In an embodiment, the therapeutic agent comprises anantibiotic. Preferably, the antibiotic comprises ciprofloxacin. Othertherapeutic agents known in the art to be effective against infectiouspathogens may also be used. Thus, other anti-infectious agents that maybe used include, but are not limited to, antiviral agents, anti-fungalagents, and compounds known to prevent infection of the eye by othermicroorganisms, such as protozoa, and the like.

In an embodiment, the therapeutic agent of the compounds and thecompositions of the present invention is known to be effective fortreatment of macular or retinal disease. For example, the therapeuticagent may comprise a compound used to treat retinoblastoma. In anotherembodiment, the therapeutic agent comprises a compound used to treatcystoid macular edema (CME). Alternatively and/or additionally, thetherapeutic agent may be a compound effective for treatment of exudativeage-related macular degeneration (AMD). In another embodiment, thetherapeutic agent is used to treat diabetic retinopathy. Still inanother embodiment, the therapeutic agent may be effective for treatmentof diabetic macular edema. The therapeutic agent may also be used totreat inflammatory disorders of the eye, such as CME or posterioruveitis.

Preferably, the prodrug of the compounds and the compositions of thepresent invention facilitates delivery of the therapeutic agent to themacula. Once in the macula, the therapeutic agent must be released fromthe carotenoid carrier for uptake and delivery into the cell. Thus, inan embodiment, the linkage between the therapeutic agent and thecarotenoid carrier comprises a biologically cleavable bond.

In an embodiment, the therapeutic agent is directly linked to thecarotenoid to form a bipartate structure. In the bipartate approach, adrug is linked directly to a carrier molecule through a chemical bond.For example, in an embodiment, the linkage between the therapeutic agentand the carotenoid carrier comprises an ester bond formed bycondensation (with loss of water) of a xanthophyll and the drug to forma bipartate compound. Depending upon the active group in the therapeuticagent, other direct bonds (e.g., carbon-carbon bonds; carbonate bonds,ether linkages) may be formed.

Alternatively, a spacer molecule is used to link the therapeutic agentto a carotenoid carrier to form a tripartate structure. In thetripartate approach, the drug is linked to a spacer, which in turn islinked to a carrier molecule. The spacer can be used to connect twomolecules that cannot be connected directly for chemical (e.g.,reactivity of active groups) or physical (e.g., steric hindrance)reasons. The spacer may be used to modify the chemical and/or physicalproperties of the system (e.g., solubility, chemical stability,enzymatic stability), or to add new properties to the system (e.g.,susceptibility to metabolism by a different enzyme). The tripartateapproach, however, may require that two bonds are cleaved to release thedrug.

For example, the linkage between the therapeutic agent and the carotenodmay comprise an amino acid spacer to form a tripartate compound. In anembodiment, the linkage between the therapeutic agent and the carotenoidmay comprise a dicarboxylic amino acid spacer. The linkage between thetherapeutic agent and the carotenod may also comprise a carbonatespacer. Thus, to form a tripartate compound, bonds linking thetherapeutic agent to the xanthopyll may comprise a carbonate bond, anester bond, or an amide bond.

In another embodiment, the present invention comprises a method for thetreatment of a disorder of the eye comprising linking a therapeuticagent to a carotenoid to create a prodrug, and administering apharmaceutically effective amount of the prodrug to an individual inneed of treatment, wherein a pharmaceutically effective amount of theprodrug comprises an amount sufficient to ameliorate, prevent, or curethe eye disorder. In an embodiment, the carotenoid comprises axanthophyll carotenoid.

Preferably, the carotenoid acts as a carrier to facilitate delivery ofthe therapeutic agent to the retina and or the macula. In an embodimentof the method, the xanthophyll comprises lutein. Alternatively, thexanthophyll may comprise zeaxanthin. Other xanthophylls known in theart, such as oxidation products of lutein and zeaxanthin, may be used.Thus, other xanthophyll compounds that may be used include, but are notlimited to, 3′-epilutein, meso-zeaxanthin, 3-hydroxy-beta,epsilon-caroten-3′-one, epsilon-lycopenes, or 5-Z-lycopenes.

In an embodiment of the methods of the present invention, thetherapeutic agent comprises an anti-angiogenic agent. Preferably, theanti-angiogenic agent comprises anecortave acetate, anti-VEGF aptamer,AMD-FAB, or protein kinase c inhibitor. Other anti-angiogenic agentsknown in the art may be used. Thus, other anti-angiogenic agents thatmay be used include, but are not limited to, steroids and angiostaticsteroids, metalloproteinase inhibitors, and interferons.

In another embodiment of the methods of the present invention, thetherapeutic agent comprises an anti-neoplastic agent. Preferably, theanti-neoplastic agent comprises etoposide or vincristine, which areanti-neoplastic agents used to treat retinoblastoma. Otheranti-neoplastic agents known in the art may also be used.

In yet another embodiment of the methods of the present invention, thetherapeutic agent comprises an anti-inflammatory agent. Preferably, theanti-inflammatory agent comprises a steroid or a non-steroidalanti-inflammatory drug (NSAID). More preferably, the anti-inflammatoryagent comprises ketorolac, triamcinolone or fluocinolone. Otheranti-inflammatory agents known to be effective for treating inflammationof the eye may be also be used. Thus, other anti-inflammatory agentsthat may be used include, but are not limited to, interleukins,anti-leukotriene agents, cyclooxygenase inhibitors, and prostaglandininhibitors.

In yet another embodiment, the therapeutic agent comprises anantibiotic. Preferably, the antibiotic comprises ciprofloxacin. Othertherapeutic agents known in the art to be effective against infectiouspathogens may also be used. Thus, other therapeutic agents that may beused include, but are not limited to, antiviral agents and agents toprevent infection of the eye by protozoa, fungi or other microorganisms.

In an embodiment of the method, the therapeutic agent is known to beeffective for treatment of macular or retinal disease. For example, thetherapeutic agent may comprise a compound used to treat retinoblastoma.In another embodiment, the therapeutic agent comprises a compound usedto treat cystoid macular edema (CME). Alternatively and/or additionally,the therapeutic agent may be a compound effective for treatment ofexudative age-related macular degeneration (AMD). In another embodiment,the therapeutic agent is used to treat diabetic retinopathy. Still inanother embodiment, the therapeutic agent may be effective for treatmentof diabetic macular edema. The therapeutic agent may also be a compoundused to treat inflammatory disorders of the eye, such as CME orposterior uveitis.

Preferably, the prodrug facilitates delivery of the therapeutic agent tothe macula. Once in the macula, the therapeutic agent must be releasedfrom the carotenoid carrier for uptake and delivery into the cell. Thus,in an embodiment of the method, the linkage between the therapeuticagent and the carotenoid carrier comprises a biologically cleavablebond.

In an embodiment of the method, the therapeutic agent is directly linkedto a carotenoid to form a bipartate structure. In the bipartateapproach, a drug is linked directly to a carrier molecule through achemical bond. For example, in an embodiment, the linkage between thetherapeutic agent and the carotenoid carrier comprises an ester bondformed by condensation (with the loss of water) of a xanthophyll and thedrug to form a bipartate compound. Depending upon the active group inthe therapeutic agent, other direct bonds (e.g., carbon-carbon bonds;carbonate bonds, ether linkages) may be formed.

Alternatively, a spacer molecule is used to link the therapeutic agentto a carotenoid to form a tripartate structure. In the tripartateapproach, the drug is linked to a spacer, which in turn is linked to acarrier molecule. The spacer can be used to connect two molecules thatcannot be connected directly for chemical (e.g., reactivity of activegroups) or physical (e.g., steric hindrance) reasons. The spacer may beused to modify the chemical and/or physical properties of the system(e.g., solubility, chemical stability, enzymatic stability), or to addnew properties to the system (e.g., susceptibility to metabolism by adifferent enzyme). The tripartate approach, however, may require thattwo bonds are cleaved to release the drug.

For example, the linkage between the therapeutic agent and thecarotenoid may comprise an amino acid spacer to form a tripartatecompound. In an embodiment, the linkage between the therapeutic agentand the carotenoid may comprise a dicarboxylic amino acid spacer. Thelinkage between the therapeutic agent and the carotenoid may alsocomprise a carbonate spacer. Thus, to form a tripartate compound, bondslinking the therapeutic agent to the xanthopyll may comprise a carbonatebond, an ester bond, or an amide bond.

Preferably, the prodrugs of the present invention may be administered bya variety of routes. For example, the prodrug may be administeredsystemically. Thus, in an embodiment, administration is intravenous.Alternatively, administration may be intramuscular. In anotherembodiment, administration is subcutaneous. In yet another embodiment,the drug is administered topically, to the surface of the eye. In yetanother embodiment, administration is oral. Also, in an embodiment,administration of the prodrugs of the present invention may comprisealtering of the diet to increase uptake of the prodrug.

Preferably, the compounds, compositions, and methods of the presentinvention allow for increased uptake of the therapeutic agent in the eyeas compared to uptake of the therapeutic agent when the carotenoidcarrier is not used. Thus, the doses for each prodrug may depend uponthe nature of the therapeutic agent, as well as the nature of thecarotenoid carrier. In an embodiment, the prodrug is administered in adosage amount that ranges from 50 mg/kg q.d. (i.e., per day) to 0.01mg/kg q.d. In another embodiment, the prodrug is administered in adosage amount that ranges from 5 mg/kg q.d. to 0.1 mg/kg q.d.

In yet another embodiment, the present invention comprises a kit for thetreatment of an eye disorder comprising:

(a) a prodrug compound comprising a therapeutic agent linked to acarotenoid;

(b) a pharmaceutically acceptable carrier, and

(c) instructions to dispense a pharmaceutically effective amount of theprodrug in the pharmaceutically acceptable carrier to an individual inneed thereof, wherein a pharmaceutically effective amount of the prodrugcomprises an amount sufficient to ameliorate, prevent, or cure an eyedisorder in the individual. In an embodiment, the carotenoid comprises axanthophyll carotenoid.

In an embodiment, the methods and compounds of the present invention mayalso used to treat diseases of other tissues known to concentratecarotenoids. For example, the compounds may also be used to treatdiseases of the liver or fat tissue.

Thus, in another embodiment, the present invention comprises a compoundfor the treatment of a disorder in a tissue type that is known toconcentrate carotenoids, comprising a therapeutic agent linked to acarotenoid. In an embodiment, the carotenoid comprises a xanthophyll.Also in an embodiment, the tissue treated comprises eye tissue, livertissue, or fat tissue. The therapeutic agent may be almost any agentthat is used to treat the disease or disorder in question and that canbe linked to a carotenoid carrier. Thus, in an embodiment, thetherapeutic agent comprises an anti-angiogenic agent, an anti-neoplasticagent, an anti-inflammatory agent, a non-steroidal anti-inflammatorydrug (NSAID), a steroid, an antibiotic, an anti-protozoan agent, or anantiviral agent.

In yet another embodiment, the present invention comprises a method forthe treatment of a disease comprising linking a therapeutic agent to acompound which is known to be naturally concentrated in a tissueaffected by, or that is causing, the disease to create a prodrug, andadministering a pharmaceutically effective amount of the prodrug to anindividual in need of treatment, wherein a pharmaceutically effectiveamount of the prodrug comprises an amount sufficient to ameliorate,prevent, or cure the disease.

In an embodiment, the compound which is naturally concentrated in thetissue affected by, or that is causing, the disease comprises acarotenoid. Preferably, the carotenoid comprises a xanthophyll. Also inan embodiment, the tissue treated comprises eye tissue, liver tissue, orfat tissue. The therapeutic agent may be almost any agent that is usedto treat the disease or disorder in question and that can be linked tothe selected carrier (e.g., a carotenoid). Thus, in an embodiment, thetherapeutic agent comprises an anti-angiogenic agent, an anti-neoplasticagent, an anti-inflammatory agent, a non-steroidal anti-inflammatorydrug (NSAID), a steroid, an antibiotic, an anti-protozoan agent, or anantiviral agent.

Thus, embodiments of the present invention utilize the ability oftissues, such as the retina and the macula, to concentrate carotenoidsas a means to enhance accumulation of therapeutic drugs in the thattissue. Lutein and zeaxanthin are members of the xanthophyll carotenoidfamily of compounds, which are characterized by a C₄₀H₅₆ isoprenoidbackbone. Carotenoids are synthesized by plants and microorganisms, inwhich they have pigmentation and photoprotective roles. Humans have alimited ability to synthesize carotenoids and thus, obtain approximately40 of these compounds from dietary sources and incorporate them intotissues, including serum, liver, and fat.

When carotenoids contain one or more oxygen atoms, as in lutein andzeaxanthin, they are known as xanthophylls. Lutein is relativelyabundant in the human diet, and is derived from such dark green, leafyvegetables as spinach, kale, and broccoli. Zeaxanthin, found at lowerblood levels, is derived from corn, peaches, citrus fruits and melons(Goodwin, T. W., Ann. Rev. Nutr., 1986, 6:273-97; Sommerburg, O., etal., Br. J. Ophthalmol., 1998, 82:907-910; Bone, R. A., et al., AcademicPress, 2000, 43:239-245; Bone, R. A., et al., Invest. Ophthalmol. Vis.Sci., 1993, 34:2033-2040; Toyoda, Y., et al., Invest. Ophthalmol. Vis.Sci., 2002, 43:1210-1221). FIG. 1 displays the chemical structures oflutein (Panel A) and zeaxanthin (Panel B).

The yellow coloration of the human macula, which is the central regionof the retina used for fine, detailed vision, is derived from a mixtureof lutein and zeaxanthin (Bone, R. A., et al., Vision Res., 1985,25:1531-1535; Bone, R. A., et al., Acad. Press, 1997, 44:211-218). Sincexanthophylls display anti-oxidant properties in vitro, they arehypothesized to scavenge free radicals and absorb short-wavelength lightwithin the eye, preventing oxidative tissue injury (Sujak, A., et al.,Arch. Biochem. and Biophys., 1999; 371:301-307; Schalch, W. In FreeRadicals and Aging, 1992, pp. 280-298; Beatty, S., et al., Surv.Opthalmol., 2000, 45:115-134).

Interestingly, it has been reported that individuals with a high dietaryintake of lutein and zeaxanthin have lower rates of age-related maculardegeneration (AMD), the leading cause of legal blindness in the elderly(Bone, R. A., et al., Academic Press, 2000, 43:239-245; Beatty, S.,Invest. Ophthalmol. Vis. Sci., 2001, 42: 439-446; Snodderly, D. M., Am.J. Clin. Nutr., 1995, 62 (suppl.):1448S-61S; Mares-Perlman, J. A., etal., Am. J. Epidemiol., 2001, 153:424-32; Seddon, J. M., et al., JAMA,1994, 272:1413-1420; Landrum, J. T., et al., Adv. Pharmacol., 1997,38:537-56). In response, lutein and zeaxanthin supplementation has beenproposed as an untested treatment for AMD, resulting in heavy marketingby the vitamin and nutrition supplement industry (Pratt, S., J. Am.Optom. Assoc., 1999, 70:39-47; Sommerburg, O., et al., Br. J.Ophthalmol., 1998, 82:907-910; Jampol, L. M., et al., JAMA, 2001,286:2466-2468). Also, oral supplementation of dietary xanthophyllsources has been experimentally shown to increase the concentration ofmacular pigment, though the precise mechanism for this accumulationremains elusive (Toyoda, Y., et al., Invest. Ophthalmol. Vis. Sci.,2002, 43:1210-1221; Hammond, B. R., et al. Exp. Eye Res., 1996,62:293-297; Landrum, J. T., et al., Exp. Eye Res., 1997, 64:311-316;Hammond, B. R., et al., Invest. Ophthalmol. Vis. Sci., 1997,38:1795-1801; Berendschot, Tos T. J. M., et al., Invest. Ophthalmol.Vis. Sci., 2000, 41:3322-3326; Bernstein, P. S., et al. Invest.Ophthalmol. Vis. Sci., 1997, 38:167-175; Leung, I. Y. F., et al.,Invest. Ophthalmol. Vis. Sci., 2001, 42: 466-471).

Thus, although the precise mechanism for xanthophyll accumulation in theretina remains obscure, there is evidence that xanthophyll compounds maybe accumulated in the macula region of the eye. Embodiments of thepresent invention use xanthophyll carotenoids, such as lutein andzeaxanthin, as carrier molecules for medications currently used in thetreatment of diseases of the eye to promote delivery and accumulation ofthe medication in the eye. By chemically bonding the xanthophyll carrierto the therapeutic compound of choice, the xanthophyll molecule providesa greater concentration of the medication within the macula than thatachieved by the medication alone.

The compounds and methods of the present invention may be used to treata wide variety of diseases of the eye. For example, diabetic retinopathyand the exudative form of AMD are characterized by neovascularization,in which the formation of abnormal blood vessels may lead to hemorrhage,retinal detachment and ultimately, legal blindness. Experimentalevidence has implicated two molecules, vascular endothelial growthfactor (VEGF) and protein kinase c, as key contributors to this process.Though conventional therapy emphasizes surgical intervention throughlaser photocoagulation, retinal cryotherapy, or photodynamic therapy,experimental, anti-angiogenic compounds, including drugs aimed atinhibiting VEGF and protein kinase c, have recently been developed andare now being tested in humans with encouraging results (Aiello, L. P.,et al., N. Engl. J. Med, 1994, 331:1480; Aiello, L. P., et al., Arch.Ophthalmol., 1995, 113:1538-1544; Kliffen, M., et al., Br. J.Ophthalmol., 1997, 81:154-62; Seo, M. S., et al., Amer. J. Pathology,1999, 154:1743-1753; Shih, S. C. et al., J. Biol. Chem., 1999,274:15407-14; Yoshiji, H., et al., Cancer Res., 1999, 59:4413-4418).Steroids are also being evaluated for the treatment of exudative AMD.

Thus, in an embodiment, the prodrug of the present invention comprisesan anti-angiogenic agent conjugated to a xanthophyll carotenoid carrier.In an embodiment, the anti-angiogenic agent linked to the xanthophyllcarrier comprises anecortave acetate, anti-VEGF aptamer (and otheranti-VEGF agents), AMD-FAB, or protein kinase c inhibitor. Linking suchagents to a xanthophyll carotenoid carrier such as lutein or zeaxanthincan increase retinal levels of these agents compared to the intravitrealor intraorbital delivery routes currently employed. In addition,xanthophyll-mediated drug delivery would avoid the complicationsassociated with invasive methods.

In an embodiment, the compounds, compositions, and methods of thepresent invention are effective for the treatment of retinoblastoma.Chemotherapy is starting to replace external beam irradiation andenucleation as the primary therapy for retinoblastoma, with radiotherapyand enucleation reserved for disease recalcitrant to chemotherapy. Forexample, retinoblastoma may treated with monthly cycles of systemicchemotherapy using drugs such as carboplatin, etoposide and/orvincristine. Laser photocoagulation with hyperthermia (transpupillarythermotherapy) and/or retinal cryopexy may also be used as adjuvanttherapies to chemoreduction. Although such procedures achieve a highsurvival rate, chemoreduction therapy routinely produces a variety oftransient side effects, ranging from nausea and vomiting, to increasedsusceptibility to infectious diseases (Lumbroso, L., et al.,Ophthalmology, 2002, 109:1130-1136). More significantly, the long-termside effects of chemotherapy in children afflicted with retinoblastoma,who are genetically susceptible to subsequent, non-ocular cancers, areunknown.

Thus, in an embodiment of the present invention, linking ananti-neoplastic drug to a xanthophyll carrier enables higher levels ofthe drug to be achieved in the retina with lower doses of theanti-neoplastic agent than currently used. Preferably, theanti-neoplastic agent comprises etoposide or vincristine, which areanti-neoplastic agents used to treat retinoblastoma. Otheranti-neoplastic agents known in the art may also be used. This increasedspecificity associated with xanthophyll promoted delivery of theanti-neoplastic agent to the eye can minimize systemic side effects andlead to enhanced drug efficacy, improved visual outcomes, and a greatersurvival rate.

Additionally, retinal inflammation, arising from surgical manipulation,infection, trauma, or immune-mediated processes, is an eye disorder thatmay benefit from xanthophyll-linked medications. For example, cystoidmacular edema (CME) is the collection of exudative fluid pockets withinthe layers of the retina. CME is a prevalent cause of visual lossfollowing cataract extraction—a common surgery performed worldwide.Although the pathogenesis of CME remains uncertain, experimental andclinical evidence implicates prostaglandin-mediated inflammation.Prostaglandins are molecules derived from arachidonic acid, aphospholipid present in cell membranes. Once released, as for exampledue to the physiological response associated with surgical manipulationof the eye, prostaglandins can lead to increased microvascularpermeability, breakdown of the blood-retinal barrier, and CME. Currentfirst-line therapy for CME includes topical NSAIDs, which inhibitprostaglandin formation from arachidonic acid; or steroids, whichdirectly block prostaglandin activity. In refractory cases, however,invasive delivery methods (such as retrobulbar injections) are used(Flach, A. J. et al., Am. J. Ophthalmol., 1991, 112:514; Flach, A. J. etal., Am. J. Ophthalmol., 1987, 103:479; Fung, W. E., Ophthalmology,1985, 92:1102; Blair, N. P., et al., In Principles and Practice ofOphthalmology, W B Saunders, 2000, pp. 2080-2088; Guex-Crosier, Y., Doc.Ophthalmol., 1999, 97:297-309; Pendergast, S. D. et al., Am J.Ophthalmol., 1999, 128:317). Thus, in an embodiment, linking NSAIDs orsteroids to a xanthophyll carrier may enable the effective prevention ortreatment of CME in patients without resorting invasive means. Given thelarge number of cataract extractions performed annually,xanthophyll-mediated drug delivery has the potential to help thousandsof patients.

Also, ocular pathogens may be treated with xanthophyll-linkedantibiotics. For example, endophthalmitis is a blinding intraocularinfection caused by bacterial or fungal organisms. Endophthalmitis maydevelop after penetrating eye trauma or as a complication of cataractand glaucoma surgery. Treatment for endophthalmitis has typicallyemployed a regimen of intravenous antibiotics. However, evidenceprovided by the Endophthalmitis Vitrectomy Study revealed that systemicadministration of antibiotics was ineffective—presumably because of poorocular penetration (Doft, B. H., Arch. Ophthalmol., 1991, 109:487).Instead, the study endorsed vitrectomy, an invasive surgical procedure,and intravitreal antibiotics.

Thus, in an embodiment, xanthophyll-mediated drug delivery may renderinvasive techniques unnecessary by systemically providing thetherapeutic levels of antibiotic needed to treat the infection.Preferably, the antibiotic comprises ciprofloxacin. Other therapeuticagents known in the art to be effective against infectious pathogens mayalso be used. Thus, other therapeutic agents that may be used include,but are not limited to, antiviral agents and agents to prevent infectionof the eye by protozoa, fungi or other microorganisms. In this way,xanthophyll-linked compounds specific for a variety of infectiousretinopathies including, but not limited to, toxoplasmosis,histoplasmosis, and cytomegalovirus retinitis, may be developed.

Thus, the present invention describes that a variety of drug classes maydisplay enhanced delivery to the retina through carotenoid carriers,potentially affecting the clinical outcomes of a wide range of oculardiseases. Using systemic administration route of carotenoid-linked drugswould avoid the complications associated with invasive techniques.

Development of Prodrugs for Delivery of Therapeutic Agents to the Retina

The present invention includes creating novel prodrugs that consist of acarotenoid carrier linked chemically to parent drugs with proven orexperimental efficacy in the treatment of retinal and macular diseases.Similar methods and approaches are used to prepare prodrugs comprisingappropriate carriers and therapeutic agents for the treatment of otherdiseases. A prodrug is a non-active molecule that releases an activeprincipal (parent drug) at the desired site of action, improving theparent drug's pharmacokinetic properties, such as bioavailability andstability.

FIG. 1 shows the chemical structures of (3R,3′R,6′R)-lutein (Panel A)and (3R,3′R)-zeaxanthin (Panel B) which, in accordance with anembodiment of the present invention, comprise xanthophyll carotenoidsthat act as carriers to concentrate compounds in the eye. Othercompounds, both xanthophyll-based and non-xanthophyll compounds, thatare known to be concentrated in the eye may also be used. For example,xanthophyll derivatives or chemically modified xanthophyll compoundssuch as 3′-epilutein, meso-zeaxanthin,3-hydroxy-beta,epsilon-caroten-3′-one, epsilon-lycopenes, or5-Z-lycopenes may be used. Also, in an embodiment, carotenes may beused.

As described herein, the therapeutic agent of interest may be directlylinked to the carotenoid carrier to form a bipartate structure. Aschematic showing conjugation of a therapeutic agent (A₁-COOH) with axanthophyll (X—OH) to form a bipartate prodrug of the present inventionis shown in FIG. 2A. As shown in FIG. 2A, a therapeutic agent having areactive (e.g., unblocked) carboxylic acid group (A₁-COOH) may becondensed with a xanthophyll (X—OH) in the presence of DCC(1,3-dicyclohexylcarbodiimide) to generate a prodrug having an esterbond forming a direct link between the xanthophyll carrier and thetherapeutic agent. The bond linking the carrier to the therapeutic agentmay be cleaved by cellular esterases. Depending upon the active group inthe therapeutic agent, other direct bonds (e.g., carbon-carbon bonds;carbonate bonds, ether linkages) may be formed.

Alternatively, a spacer molecule may be used to link the therapeuticagent to the carotenoid carrier to form a tripartate structure. Aschematic showing an embodiment of a tripartate prodrug of the presentinvention is shown in FIG. 2B. As shown in FIG. 2B, a therapeutic agenthaving a reactive (e.g., unblocked) hydroxyl group (A₂COCH₂OH) may becondensed with an amino acid ester (e.g.ω-methyl-N-carbobenzyloxy-aspartic or glutamic acid) (N-CBZ) in thepresence of DCC to result in the formation of a direct bond between thetherapeutic agent and the spacer upon loss of water. Next, a xanthophyllcarrier (X—OH) is added by selective saponification and condensation(with loss of water) in the presence of DCC. Thus, the resulting prodrugcomprises the therapeutic agent linked via an amino acid spacer to thexanthophyll.

The present invention describes the use of known and experimentaltherapeutic agents linked to carotenoid carriers for delivery to theeye. Therapeutic agents linked to carotenoid carriers may include thefollowing: (1) etoposide (commercially available from MylanPharmaceuticals, Morgantown, W.V.) and vincristine, (Oncovin®;commercially available from Eli Lilly, Indianapolis, Ind.) bothanti-neoplastic agents used to treat retinoblastoma; (2) ketorolac(commercially available from Roche Laboratories, Nutley, N.J.), an NSAIDused to treat cystoid macular edema (CME) and posterior uveitis; (3)anecortave acetate (Alcon Inc., Forth Worth, Tex.), a putativeanti-angiogenic steroid derivative for treatment of diseases thatinvolve retinal neovascularization with concomitant visual loss such asexudative AMD, diabetic retinopathy (DR), and diabetic macular edema(DME); (4) the anti-VEGF aptamer pegaptanib (MACUGEN™ or EYE001,commercially available from EyeTech, Inc., New York, N.Y.), AMD-FAB(Rhu-Fab, a humanized antibody fragment; Genentech, San Francisco,Calif.), and protein kinase c inhibitor (Eli Lilly, Indianapolis, Ind.),three putative anti-angiogenic compounds that could also be used totreat exudative AMD, DR, and DME; (5) triamcinolone and fluocinolone(both commercially available from various suppliers), steroids used totreat such inflammatory disorders as posterior uveitis and CME, as wellas exudative AMD, DR, or DME; and (6) ciprofloxacin (commerciallyavailable from Bayer Corporation, West Haven, Conn.), a fluoroquinoloneantibiotic that can be used to treat a variety of ocular infections.Xanthophyll compounds may be obtained from Hoffmann-La Roche (Nutley,N.J.). The purity of all medications and xanthophyll carriers may beverified by high-performance liquid chromatography (HPLC). Table 1describes at least some of the therapeutic agents which may used togenerate the prodrugs of the present invention.

TABLE 1 Examples of prodrugs and their potential applications PotentialProdrug Drug Category Chemical Structure of Therapeutic AgentApplication(s) lutein-etoposide, zeaxanthin- etoposide anti-neoplasticagent

retinoblastoma lutein-vincristine, zeaxanthin- vincristineanti-neoplastic agent

retinoblastoma lutein-ketorolac, zeaxanthin- ketorolac NSAID*

CME** lutein-anecortave acetate, zeaxanthin- anecortave acetate putativeanti- angiogenic steroid derivative

exudative AMD, DR, DME*** lutein-protein kinase c inhibitor, zeaxanthin-protein kinase c inhibitor putative anti- angiogenic compounds

exudative AMD, DR, DME lutein- triamcinolone, zeaxanthin- triamcinolonesteroid [see Scheme 5, below] posterior uveitis, CME, exudative AMD, DR,DME lutein- fluocinolone, zeaxanthin- fluocinolone steroid

posterior uveitis, CME, exudative AMD, DR, DME lutein- ciprofloxacin,zeaxanthin- ciprofloxacin fluoroquinolone antibiotic

endophthalmitis *NSAID = non-steroidal anti-inflammatory drug **CME =cystoid macular edema ***AMD = age-related macular degeneration; DR =diabetic retinopathy; DME = diabetic macular edema

In designing a prodrug, several factors are generally considered:Preferably, the prodrug is non-toxic and biologically inactive.Additionally, the prodrug should be resistant to serum enzymatic orchemical degradation. Also preferably, the prodrug has alipophilic-hydrophilic balance and water solubility to ensurepenetration of biological membranes. By selecting the appropriatenatural carrier, the resulting prodrug is able to use specific,intermembrane transport mechanisms to be delivered to the tissue siterequired. Once inside the targeted tissue, the parent drug is releasedfrom the prodrug by enzymatic or chemical hydrolysis of the drug-carrierbond.

As described herein, bipartate and tripartate compounds may used in thedesign of prodrugs. In the bipartate approach, the drug and carrier(e.g., carotenoid) are directly bound. In the tripartate approach, thedrug and carrier are connected by a spacer. The type of chemical bond,as well as the chemical properties of the spacer, can be used to controlthe stability and lipophilic-hydrophilic character of the prodrug(Cooperwood, J. S. et al., In Chemistry and Chemotherapy, C. K. Chu,ed., 2002, pp. 91-147).

To develop prodrugs comprising a xanthophyll carrier, several issuesregarding the proposed drug-carrier systems must be addressed. First,lutein has two non-equivalent and stereoelectronically similar OHgroups. Thus, selective monosubstitution is difficult because thereaction can yield a mixture of diastereomers. In contrast, zeaxanthinis a better candidate for monosubstitution because the C2 symmetry ofzeaxanthin only produces only one isomer.

Also, depending on the nature of the therapeutic agent, disubstitutionof the xanthophyll carrier may make the resulting compound toolipophilic and insoluble for adequate tissue adsorption and distributionto be achieved. In an embodiment, a number of hydroxyl (OH) or amine(NH₂) groups can be left free (or included in a spacer) to ensure a goodlipophilic-hydrophilic balance. In general, however, disubstitutedprodrugs are too insoluble for adequate absorption and distribution tobe achieved.

Finally, since zeaxanthin is less likely to be deposited in suchnon-retinal tissues as fat (Toyoda, Y., et al., Invest. Ophthalmol. Vis.Sci. 2002; 43:1210-1221; Johnson, E. J., et al., Am. J. Clin. Nutr.2000; 71:1555-62; Kaplan, L. A., et al., Clin. Physiol. Biochem., 1990;8:1-10), it may target the retina more effectively than lutein. Thesynthesis of a variety of prodrugs using zeaxanthin as a carrier isdescribed in schemes 1-5 shown below. Analogous methods may be used forthe preparation of prodrugs using other xanthophyll carriers.

Scheme 1. Zeaxanthin-Etoposide Prodrugs:

The anti-neoplastic agent etoposide is characterized by a polycyclicstructure in which a vicinal diol and phenolic group can befunctionalized. The acidity and reactivity of the phenolic group can beexploited to achieve stereoselectivity. However, the steric hindrancearound these groups can influence reactivity in an unpredictable way,necessitating a spacer to connect the bulky drug and xanthophyllmoieties.

The above scheme shows an example in which the etoposide diol isprotected as acetonide and the phenolic hydroxyl is functionalized withan amino acid spacer. Subsequently, the etoposide-spacer compound iscoupled, via a condensation reaction and loss of water, to zeaxanthin.Abbreviations used in the above scheme are as follows:PTSA=para-toluenesufonic acid; N-CBZ=N-carbobenzyloxy; andDCC=1,3-dicyclohexylcarbodiimide.

The use of amino acids as spacers is convenient because they arenatural, non-toxic molecules and because their presence generallyimproves the hydrophilicity of the resulting prodrug. In addition, theuse of different amino acids allows the synthesis of prodrugs withvarying hydrophilicities and resistance to enzymatic degradation. In anembodiment, these aminoacyl prodrugs may release their parent drugthrough amidases and esterases.

Scheme 2. Zeaxanthin-vincristine prodrugs: The anti-neoplastic agentvincristine is an alkaloid characterized by two quaternary groups and anindole ring. Prodrugs may be synthesized by functionalizing one of thetwo hydroxyl groups. The above scheme illustrates how zeaxanthin andvincristine can be coupled via a carbonate spacer. After blocking theamine group of vincristine, a bridging carbonyl group is added first tothe hydroxyl group(s) of vincristine and then to the hydroxyl group ofzeaxanthin by electrophilic substitution using oxalyl chloride (COCl₂).A carbonate spacer, which contains labile bonds, is appropriate becausevincristine contains moieties that are potentially unstable. Thisprodrug is designed to release its parent drug throughesterase-catalyzed hydrolysis. BOC is the t-butoxycarbonyl protectinggroup.

Scheme 3. Zeaxanthin-ketorolac prodrugs: Ketorolac is commerciallyavailable as the trimethamine salt. For the purpose of derivatization,the free acid can be used, although it is a racemate and will produce aninseparable, diastereomeric mixture when coupled directly toxanthophylls. The upper pathway of scheme 3 shows direct condensationbetween ketorolac and zeaxanthin, using either a dehydrating agent suchas DCC, or via an acyl chloride intermediate. Both methods will give themono- and disubstituted esters, however, careful control of reactionconditions should permit maximal yield of either product. In anembodiment, these ester prodrugs can release their parent drug throughesterase-catalyzed hydrolysis.

The tripartate prodrug can also be synthesized, as illustrated in thelower pathway of the scheme, through condensation with an amino acidester followed by selective saponification and coupling to zeaxanthin.These aminoacyl prodrugs should release their parent drug throughamidases and esterases.

Scheme 4. Zeaxanthin-anecortave prodrugs: Anecortave is commerciallyavailable as an acetate. The acetate group is not essential for itsactivity, however, and may be substituted with a xanthophyll carrier.Although the xanthophyll carrier may be coupled to the secondaryalcohol, substitution at the primary hydroxyl group should give a stableprodrug as the primary hydroxyl groups in both anecortave and thexanthophyll carrier are sterically hindered. To this end, a tripartateapproach, in which the spacer is either a dicarboxylic amino acid(aspartic or glutamic acid) or a carbonate group, is appropriate. Scheme4A shows formation of a zeaxanthin-amino acid-anecortave prodrug; in anembodiment, decomposition of this compound can be catalyzed by amidaseenzymes. Scheme 4B shows formation of the zeaxanthin-anecortave prodrugwith a carbonate spacer; in an embodiment, decomposition of thiscompound should be catalyzed by esterases.

Scheme 5. Zeaxanthin-triamcinolone or fluocinolone prodrugs:Triamcinolone and fluocinolone are steroids that differ only in afluorine substitution. Both of them are commercially available asacetonides or free polyalcohols. As in the case of anecortave, they canbe coupled to xanthophylls via a dicarboxylic amino acid or carbonatespacer. These two approaches are shown in schemes 5A and 5B, above.

Scheme 6. Zeaxanthin-ciprofloxacin prodrugs: Ciprofloxacin is afluoroquinolone antibiotic whose structure is characterized bycarboxylic and secondary amino groups. The carboxylic group allows thecreation of prodrugs through procedures outlined for ketorolac, above(Scheme 3). Thus, the above scheme shows the making of bipartate esterprodrugs in the upper scheme and tripartate prodrugs (with an amino acidspacer) in the lower scheme. Decomposition of these prodrugs iscatalyzed by esterase and amidase enzymes, respectively.

After synthesis, prodrugs may be tested to characterize the stability ofthe carotenoid-parent drug bond. There are several animal systems whichhave been used as models to evaluate carotenoid metabolism. For example,studies related to xanthophyll metabolism and supplementation of thediet with xanthophylls have been carried out using frogs, rainbow trout,monkeys, and Japanese quail.

Thus, in an embodiment, prodrug stability may be evaluated using a quailliver homogenate. Livers of untreated quail may be surgically removed,ground, and mixed with saline solution and the prodrug of interest. Afraction of the resulting homogenate is extracted with acetonitrile, thesupernatant dried with nitrogen gas, and a portion analyzed for prodrug,parent drug (e.g. therapeutic agent) and carotenoid levels by HPLC.Standard curves for both parent drug and prodrug stability areconstructed using low, intermediate, and high concentrations of prodrugincubated in the liver homogenate for a range of times (e.g., 0.5 hrs upto 24 hours). Stability evaluation allows for development of prodrugs(through modification of the synthetic scheme) that are be stable enoughto survive hematogenous transport to the retina while still remainingvulnerable to retinal hydrolytic enzymes.

Assessing Prodrug Delivery to the Target Tissue

In an embodiment, prodrugs may be evaluated to determine whether linkingthe therapeutic agent to a carotenoid carrier aids drug delivery to thetissue of interest (e.g., retina). For this purpose, 6 week old adultJapanese quail (Coturnix japonica), which have been shown to be a usefulanimal model in the study of carotenoid delivery to the retina, may beused (Toyoda, Y., et al., Invest. Ophthalmol. Vis. Sci., 2002,43:1210-1221).

i. Effect of a Xanthophyll-Deficient Diet on Prodrug Uptake

In an embodiment, an analysis is performed to determine whether axanthophyll-deficient diet leads to depletion of retinal xanthophyllstores, and whether a wash-out period of xanthophyll depletion precedingexogenous xanthophyll supplementation increases prodrug uptake in theeye, or other tissue of interest.

For these studies, Japanese quail (Toyoda, Y., et al., Invest.Ophthalmol. Vis. Sci., 2002, 43:1210-1221), or other model systems knownby those of skill in the art, may be used. For example, in anembodiment, 6 week old quail are divided into 2 groups, each containinga predetermined number of quartets each having two male and two femaleanimals. Group 1 receives a modified gamebird diet deficient in luteinand zeaxanthin (Purina Test Diet, Purina Mills, Richmond, Ind.) andGroup 2 receives a standard gamebird diet, with each quartet being fedtheir respective diet for a predetermined time periods. For example,quartets A-G of each group may be fed their respective diets for 0, 1,2, 4, 6, 8, and 12 weeks, respectively. The absence of xanthophyllswithin the modified food can be confirmed by HPLC.

At the conclusion of each time period, the quail are decapitated, andfor quantitative analysis of xanthophylls in the retina, the eyesquickly enucleated and placed on ice. A circumferential incision may bemade 2 to 3 mm anterior to the ocular equator, the anterior segment andlens is removed, and ice-cold phosphate buffer containing 50 μM EDTA isplaced in the eyecup. Curved iridectomy scissors are used to cut andremove the vitreous close to the inner limiting membrane of the retinaand, using intersecting cuts lateral to the pecten, the retina is peeledfrom the retinal pigment epithelium (RPE). Other tissue types may alsobe analyzed using biopsy techniques known in the art. All specimens,along with serum samples from each individual, are stored in darkness at−70° C. until analysis by HPLC (Toyoda, Y., et al., Invest. Ophthalmol.Vis. Sci., 2002, 43:1210-1221).

For interpretation of the data, a statistical analysis comparingcovariance of the heterogeneity of the slopes over time for the 2 groups(i.e., low and normal xanthophyll diet) is evaluated. If the slopes arenot different, than a wash-out period with a xanthophyll-deficient dietis unnecessary. If the slopes are different, post hoc within timet-tests (adjusted for the multiple comparison) may be used to determinethe time point at which the groups become different to define theappropriate wash-out period.

ii. Drug Delivery Route

Also, in an embodiment of the present invention, the preferred deliveryroute for the prodrug of interest may be determined. For example, in anembodiment, topical, intravenous, and intramuscular routes ofadministration are compared. The analysis also allows for an assessmentof the efficacy of interstitial enzymes to hydrolyze the bond(s) betweenparent drug (i.e., therapeutic agent) and the carotenoid carrier.Additionally, the analysis may allow for assessment of any tissuenecrosis that may result as part of the administration of the drug, suchas necrosis resulting from the use of injection needles, subcutaneouspumps, or from the drug itself.

For this assessment, 6 week old quail may be divided into groupscorresponding to the different modes of administration. For example, inan embodiment, there may be three test groups: (1) intravenousadministration, (2) intramuscular administration, and (3) subcutaneousadministration, with each group containing 2 quartets each having twomale and two female birds, such that the first quartet of each groupreceives the parent drug (e.g., either ketorolac or triamcinolone) whilethe second quartet receives the corresponding prodrug (e.g., ketorolaclinked to zeaxanthin). Subcutaneous administration of the drug maycomprise the use of subcutaneous pumps (Alzet Osmotic Pumps; DurectCorporation, Cupertineo, Calif.). Dosages of parent drugs may range fromabout 2 mg/kg q.d. (e.g., ketorolac) to about 0.2 mg/kg q.d. (e.g.,triamcinolone). Prodrug dosages are the molar equivalents of the drugdoses.

The test subjects may receive either a xanthophyll-deficient diet or astandard diet as required to maximize xanthophyll uptake. After apredetermined time of prodrug/drug administration (e.g. 12 weeks for thequail assay), the animals are sacrificed, and serum and retinal tissue(and/or other tissue of interest) harvested for HPLC analysis ofprodrug, drug, and xanthophyll levels. For data interpretation, atwo-way analysis of variance may be used to determine differences (ifany) between the different drug groups, the different modes of delivery,and any interaction between drug type and mode of delivery.

iii. Xanthophyll Levels in Retina

In another embodiment of the present invention, the time required forxanthophyll accumulation in the retina (or other tissue of interest) isdetermined. In an embodiment, 6 week old quail are divided into 2groups, each containing 7 quartets each having two male and two femalebirds. Group 1 receives a lutein- and zeaxanthin-deficient diet whileGroup 2 receives a standard gamebird diet. After an optimum wash-outperiod, quartets A-G of each group receive a xanthophyll carrier (i.e.,no therapeutic agent) using a preferred administration method (e.g.,intravenous, subcutaneous, or intramuscular) and at a dosage equivalentto the molar quantity of the recommended dosage for the therapeuticagent of interest for increasing times (e.g, 0 day, 1 day, 1 week, 2weeks, 4 weeks, 8 weeks, and 12 weeks).

At the conclusion of each time period, the test animals are sacrificed,serum, retina, and/or other tissue specimens collected, and tissuesamples analyzed by HPLC for lutein and zeaxanthin content to determinethe administration time necessary to produce measurable differences inxanthophyll levels for the tissue of interest. In an embodiment, thestatistical analysis comprises an analysis of covariance ofheterogeneity of the slopes across time for the two groups, with posthoc within time t-tests (adjusted for multiple comparisons) to determinewhen the difference occurs.

iv. Morbidity/Mortality Analysis

In an embodiment, the carotenoid conjugates of the present invention areevaluated for any potential toxicity. The therapeutic efficacy ofendogenously active compounds can be determined by standardpharmaceutical procedures in cell culture or experimental animals usingprocedures known in the art. The dose ratio between toxic andtherapeutic effects is the therapeutic index and may be expressed asLD₅₀/ED₅₀, wherein LD₅₀ is understood to represent the dose which istoxic to 50% of the subjects and ED₅₀ is understood to represent thedose which is effective in 50% of the subjects. Generally, compoundswhich exhibit large therapeutic indices are preferred.

For example, in an embodiment, toxicity is evaluated using the quailmodel. At age 6 weeks, quail may be divided into 2 groups, eachcontaining 7 quartets (e.g. A-G) of two males and 2 females to test thetoxicity of parent drugs and prodrugs. Both groups may receive a dietdetermined to maximize xanthophyll accumulation in the retina or othertissue of interest. For example, quartets A-G of Group 1 receiveincreasing amounts (e.g., 0.1×, 0.3×, 1×, 3×, 10×, 30×, and 100× of therecommended daily intravenous dose) of the therapeutic agent of interest(e.g., ketorolac or triamcinolone). Quartets A-G of Group 2 receivesmolar equivalent doses of the corresponding prodrugs (e.g., ketorolaclinked to zeaxanthin). For example, in an embodiment, a recommended dose(1×) of ketorolac is 2 mg/kg q.d., and a recommended dose (1×) oftriamcinolone is 0.2 mg/kg q.d. Morbidity is assessed by observing quailfor behavioral changes, feather loss, the appearance of new lesions,etc. The lethal dose is defined as the point at which mortality isgreater than, or equal to, a predetermined amount (e.g., 25%) of theindividuals within a quartet. After the period of time required tomaximize xanthophyll accumulation in the retina (or other tissue ofinterest), the quail are sacrificed, and tissue samples (e.g., serum andretina) may be analyzed by HPLC for parent drug, prodrug, andxanthophyll concentrations. Values obtained may be used to construct adose-response curve, enabling selection of the optimal safe drug andprodrug dosage. Analysis of serum data will also allow a determinationof the degree of prodrug hydrolysis prior to tissue deposition.

Analysis of ProDrug-Targeting

In another embodiment of the present invention, the ability of theprodrugs of the present invention to target the therapeutic agent to theretina and/or other tissue of interest is evaluated. For example, inthese experiments six-week-old quail may be fed either a lutein- andzeaxanthin-deficient diet, or a regular diet as determined to maximizexanthophyll uptake in the retina. This period (i.e., the period duringwhich the quail receive a xanthophyll-deficient diet without any drugs),is termed the “wash-out” period. After completion of a pre-set wash-outperiod, quail may be divided into 5 groups of four males and fourfemales per group. Group C (control) receives sterile 0.9% saline, GroupL (linked xanthophyll and drug) receives daily doses of the prodrugs ofinterest, Group D (parent drug alone) receives daily doses of the parentdrug only, Group X (xanthophyll alone) receives daily doses of unlinkedxanthophyll, and Group U (unlinked xanthophyll and unlinked drug)receives daily doses of unlinked xanthophyll and unlinked parent drug.For this evaluation, drugs and xanthophylls may be administered for apredetermined time period and using a method of administrationdetermined to be optimal. Groups L, D, and U receive equivalent doses ofmedications. Groups X and U receive a dosage of xanthophyll carrierequal to the molar quantity of xanthophyll administered to Group L. Toavoid any errors in group identification, each quail receives acolor-coded leg band.

At the conclusion of the experimental period, quail are sacrificed andtheir retinas and other tissues of interest removed. Samples of retina,as well as serum, liver, and fat (i.e., tissues known to accumulatexanthophylls) (Toyoda, Y., et al., Invest. Ophthalmol. Vis. Sci., 2002,43:1210-1221), along with brain, heart, and kidney, are harvested todetermine the specificity of drug targeting. Tissue samples may beanalyzed by HPLC for xanthophyll, prodrug, and parent drug content.Table 2 summarizes the experimental groups used for drug targetinganalysis.

TABLE 2 Xanthophyll-Drug Regimens and Measured Compounds Group ToReceive Measured Compounds* C (control) 0.9% saline xanthophyll,prodrug, and parent drug L (linked prodrug xanthophyll, prodrug,xanthophyll and parent drug and drug) D (conventional conventionalxanthophyll, prodrug, drug, alone) (parent) drug and parent drug X(xanthophyll, xanthophyll xanthophyll, prodrug, alone) and parent drug U(unlinked unlinked xanthophyll, prodrug, xanthophyll xanthophyll, andparent drug and unlinked unlinked drug drug) *Measured compounds definethe compounds to be assayed: for example, in the control, the measuredprodrug (and presumably the parent drug) serves as a negative control,and the measured xanthophylls provides a measure of endogenousxanthophyll levels.

Dietary supplementation of zeaxanthin has been shown to produce a 4-foldincrease in zeaxanthin concentration in quail fed a carotenoid-deficientdiet (Toyoda, Y., et al., Invest. Ophthalmol. Vis. Sci., 2002,43:1210-1221). Although the absolute concentration differed between thetwo sexes, the magnitude of the increase was equivalent. Thus, for theprodrugs of the present invention, Groups L, X, and U should exhibit anincrease in retinal xanthophyll concentration over xanthophyll controlGroups C and D. Thus, in an embodiment, a sample size of 8 quail pergroup (4 male and 4 female) provides adequate power (power>80%) todetect differences of this magnitude when alpha=0.05.

For this analysis, the quantity of unaltered drugs and xanthophylls isthe variable of interest. In order to determine xanthophyll and drugconcentration differences between the different experimental groups, aone-way analysis of variance (ANOVA) can be performed within each regionof measurement (retina, serum, liver, fat, brain, heart, and kidney). Ifthe F-test for a group is significant, a series of preplanned contraststo measure group differences of interest can then be used. The set ofthree orthogonal contrasts to determine differences in measured drugquantity is Groups L vs. D, Groups L vs. U, and Groups D vs. U. The setof 3 orthogonal contrasts to determine xanthophyll quantity differencesis Groups L vs. X, Groups L vs. U, and Groups X vs. U. In addition, aDunnett's test for all variables to establish differences between GroupsL, D, X, and U and the placebo control group is used.

A multivariate analysis of variance (MANOVA) may be used to determinedrug or xanthophyll concentration differences between the five groups(e.g., C, L, D, X, and U) and region of measurement, with adjustmentsfor post hoc multiple comparisons of significant effects and investigatecorrelations of measurements between regions.

In addition, the normality of the drug and xanthophyll concentrationmeasurements is assessed. If normality cannot be assumed, the analysisis performed on the rank or log transformed data or by appropriatenonparametric procedures. Results are reported as mean and SD for normaldata or median and interquartile range for non-normal data.

Therapeutics

The invention contemplates methods of administration which are wellknown in the art. For example, in an embodiment, administration of thecompound is systemic, as for example by parenteral administration, usingintramuscular, subcutaneous, intravenous, or intra-arterial routes. Inyet another embodiment, administration is topical to the eye, as forexample using eye drops. In another embodiment, the method ofadministration is by a transdermal patch. Also, administration mayemploy a time-release capsule. In yet another embodiment, administrationof the compound is oral or as an aerosol. In another embodiment,administration of the compound is sublingual.

Pharmaceutical formulations can be prepared by procedures known in theart. For example, the compounds can be formulated with commonexcipients, diluents, or carriers, and formed into tablets, capsules,suspensions, powders, and the like. Examples of diluents that aresuitable for systemic administration include water, saline and/orbuffered physiological solutions. Also, physiological preservatives(e.g., benzalkonium chloride), antibiotics, and compounds to adjust theosmolarity of the formulation of the solution may be included.

Other fillers and carriers which may also be employed, depending uponthe method of uptake, include the following: fillers and extenders suchas starch, sugars, mannitol, and silicic derivates; binding agents suchas carboxymethyl cellulose and other cellulose derivatives, alginates,gelatin, and polyvinyl pyrrolidone; moisturizing agents such asglycerol; disintegrating agents such as agar, calcium carbonate, andsodium bicarbonate; agents for retarding dissolution such as paraffin;resorption accelerators such as quaternary ammonium compounds; surfaceactive agents such as cetyl alcohol, glycerol monostearate; adsorptivecarriers such as kaolin and bentonite; and lubricants such as talc,calcium and magnesium stearate, and solid polyethyl glycols.

The compounds can also be formulated as elixirs or solutions forconvenient oral administration. Additionally, the compounds are wellsuited to formulation as sustained release dosage forms and the like.The formulations can be so constituted that they release the activeingredient only or preferably in a particular part of the intestinaltract, possibly over a period of time. The coatings, envelopes, andprotective matrices may be made, for example, from polymeric substancesor waxes.

In an embodiment, the dose of prodrug comprises levels of thetherapeutic agent of interest that are used pharmacologically in animalsand humans. Also preferably, the dose of prodrug in a localconcentration of therapeutic agent which ranges from 0.005 nM to 50 μM,and more preferably, from 0.05 nM to 1 μM, or even more preferably, from1 nM to 100 nM.

Also, the ability of prodrug may a function of cell division and thelength of the cell cycle. Thus, application of the prodrug may behourly, daily, or over the course of weeks. Thus, preferably, theeffective amount of the prodrug comprises from about 1 ng/kg body weightto about 200 mg/kg body weight. More preferably, the effective amount ofthe prodrug comprises from about 1 μg/kg body weight to about 50 mg/kgbody weight. Even more preferably, the effective amount of the prodrugcomprises from about 10 μg/kg body weight to about 10 mg/kg body weight.Alternatively, a continuous level of prodrug ranging from about0.05-10,000 μg/kg/hour, or more preferably, 0.5-250 μg/kg/hr, or evenmore preferably 5-50 μg/kg/hour may be employed. The actual effectiveamount will be established by dose/response assays using methodsstandard in the art. Thus, as is known to those in the art, theeffective amount will depend on bioavailability, bioactivity, andbiodegradability of the compound.

It will be understood that each of the elements described above, or twoor more together, may also find utility in applications differing fromthe types described. While the invention has been illustrated anddescribed as methods and compositions for treatment of macular andretinal disease, it is not intended to be limited to the details shown,since various modifications and substitutions can be made withoutdeparting in any way from the spirit of the present invention. As such,further modifications and equivalents of the invention herein disclosedmay occur to persons skilled in the art using no more than routineexperimentation, and all such modifications and equivalents are believedto be within the spirit and scope of the invention as described herein.

1. A compound for the treatment of an eye disorder in a patient comprising a therapeutic agent chemically linked to a carotenoid, wherein said therapeutic agent is an anti-angiogenic compound selected from the group consisting of anecortave acetate, pegaptanib (EYE001), Rhu-fab and a protein kinase C inhibitor according to the chemical structure:

and wherein the carotenoid acts as a carrier to facilitate uptake of the therapeutic agent in the retina and or the macula.
 2. The compound of claim 1, wherein the carotenoid comprises a xanthophyll.
 3. The compound of claim 2, wherein the xanthophyll comprises zeaxanthin.
 4. The compound of claim 1, wherein the linkage between the therapeutic agent and the carotenoid comprises a biologically cleavable bond.
 5. The compound of claim 1, wherein a spacer molecule is used to link the therapeutic agent to the carotenoid.
 6. The compound of claim 1, wherein the linkage between the therapeutic agent and the carotenoid comprises an amino acid spacer, a carbonate spacer, an ester bond, an amide bond, or a dicarboxylic acid bond.
 7. The compound of claim 1 wherein said therapeutic agent is anecortave acetate.
 8. The compound of claim 1 wherein said therapeutic agent is pegaptanib.
 9. The compound of claim 1 wherein said therapeutic agent is Rhu-fab.
 10. The compound of claim 1 wherein said therapeutic agent is a said protein kinase C inhibitor.
 11. A composition for the treatment of an eye disorder in a patient comprising a pharmaceutically effective amount of a prodrug comprising a therapeutic agent linked to a carotenoid, wherein said therapeutic agent is an anti-angiogenic compound selected from the group consisting of anecortave acetate, pegaptanib (EYE001), Rhu-fab and a protein kinase C inhibitor compound according to the chemical structure:

and a pharmaceutically acceptable carrier, wherein a pharmaceutically effective amount of the prodrug comprises an amount sufficient to ameliorate the eye disorder.
 12. The composition of claim 11, wherein the carotenoid comprises a xanthophyll.
 13. The composition of claim 11 wherein said therapeutic agent is anecortave acetate.
 14. The composition of claim 11 wherein said therapeutic agent is pegaptanib.
 15. The composition of claim 11 wherein said therapeutic agent is Rhu-fab.
 16. The composition of claim 11 wherein said therapeutic agent is said protein kinase C inhibitor.
 17. A kit for the treatment of an eye disorder in a patient comprising: (a) a prodrug compound comprising a therapeutic agent linked to a carotenoid wherein said therapeutic agent is an anti-angiogenic agent selected from the group consisting of anecortave acetate, pegaptanib (EYE001), Rhu-fab and a protein kinase C inhibitor according to the chemical structure:

(b) a pharmaceutically acceptable carrier; and (c) instructions to dispense a pharmaceutically effective amount of the prodrug in the pharmaceutically acceptable carrier to an individual in need thereof, wherein a pharmaceutically effective amount of the prodrug comprises an amount of sufficient to ameliorate an eye disorder in the individual.
 18. The kit of claim 17, wherein the carotenoid comprises a xanthophyll.
 19. The kit of claim 17 wherein said therapeutic agent is anecortave acetate.
 20. The kit of claim 17 wherein said therapeutic agent is pegaptanib.
 21. The kit of claim 17 wherein said therapeutic agent is Rhu-fab.
 22. The kit of claim 17 wherein said therapeutic agent is said protein kinase C inhibitor. 